BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 31. GENE REARRANGEMENTS: RECOMBINATION, TRANSPOSITION, AND CLONING

31.11. Specific Eukaryotic Genes Can Be Isolated by Cloning from Total Genomic DNA Cleaved with Restriction Endonucleases

As discussed in the previous chapter, studying Eukaryotic Genomes presents formidable difficulties. A Gene 1 kb in length makes up 2.5 • 10^-4 of the E. coli genome and only 3.4 • 10^-7 of a mammalian genome. Recombinant DNA technology now makes it possible to introduce a eukaryotic gene into E. coli, greatly simplifying the task. The experiment begins with the partial Digestion of eukaryotic genomic DNA to yield random fragments with an average length of approximately 20 kb (Fig. 31.25). Synthetic linkers are attached to the ends of these fragments, cohesive ends are formed, and they are then joined to a vector, such as bacteriophage lambda DNA. Packaging the DNA into virions in vitro selects for recombinant DNA molecules containing large inserts. These recombinant phages are then used to infect E. coli Cells. The result is a lysate containing fragments of eukaryotic DNA enclosed in phages and amplified about a million-fold. This lysate constitutes a cloned eukaryotic DNA library.

Class="center">Fig. 31.25. Strategy for cloning a specific eukaryotic gene starting from the Cleavage of total genomic DNA

This library can then be screened to identify phage clones containing the desired eukaryotic gene. Calculations show that only about 1 in 180,000 clones will contain a unique eukaryotic gene. Therefore, a very rapid and efficient screening Procedure is required. This procedure is based on Hybridization. The presence of a specific DNA sequence in a single bacteriophage plaque can be detected using a radioactive complementary DNA or RNA molecule as a hybridization probe. The binding of this probe can be detected by autoradiography. Thus, 1 million clones can be screened in a single day. In short, a clone corresponding to a specific eukaryotic gene can be readily identified and isolated, provided that an RNA transcribed from it is available in a more or less pure form.

31.12. Eukaryotic Genes Can Be Transcribed in Bacterial Cells

Recombinant DNA molecules containing bacterial genes are frequently expressed in E. coli cells. For example, cloning the E. coli Tryptophan Operon within the plasmid vector ColE1 leads to The production of large amounts of the five biosynthetic Enzymes encoded by this operon. The amount of these enzymes is roughly 20 times higher than in a normal E. coli Cell, because the recombinant plasmid is present in multiple copies. DNA from Yeast, a simple eukaryotic Organism, can also be expressed in Bacteria. In one study, an E. coli mutant requiring Histidine—due to a deficiency in imidazoleglycerol-phosphate dehydratase—was used as the host cell. Upon infection of this mutant with phage lambda carrying a fragment of yeast DNA, several bacterial clones appeared that no longer required exogenous histidine. The inserted yeast DNA fragment carried the missing gene, which was expressed using the METABOLISM/31.html">Transcription and Translation machinery of the bacterial cell.

Can mammalian genes be expressed in bacteria? To answer this question, the rat Insulin gene was introduced into E. coli cells (Fig. 31.26). The starting point for this study was an insulinoma, a pancreatic tumor that secretes large amounts of insulin. This tumor is rich in preproinsulin mRNA, the precursor to the active hormone (Section 35.9). Double-stranded complementary DNA (cDNA) was synthesized from this mRNA using reverse transcription and then incorporated into a plasmid vector. Why was cDNA rather than genomic DNA introduced into E. coli? As noted previously, Many eukaryotic genes contain intervening sequences that are spliced out of primary transcripts (Section 29.16). Because bacteria are presumably unable to remove these sequences, it is desirable to transform them with a DNA segment complementary to mature mRNA. Indeed, it was found that several bacterial clones transformed with the insulin cDNA synthesized small amounts of an insulin precursor (about 100 copies per cell). Another significant result was obtained recently: the DNA sequence encoding chicken egg Ovalbumin was successfully expressed in E. coli cells. About 1.5% of the protein synthesized in such transformed bacteria consisted of complete ovalbumin molecules (molecular mass 43 kDa). Clearly, eukaryotic protein genes can be expressed in bacteria.

cDNA — complementary DNA synthesized by Reverse Transcriptase using an RNA template.

Fig. 31.26. Synthesis of the insulin precursor proinsulin in transformed E. coli cells

31.13. A Chemically Synthesized Gene for the Peptide Hormone Somatostatin Is Expressed in E. coli Cells

Recent advances in the Chemical synthesis of predetermined DNA sequences have greatly expanded the scope and power of recombinant DNA technology. Genes with virtually any desired nucleotide sequence can be synthesized de novo and inserted into a vector for Introduction into E. coli. A prime example of this approach is the synthesis of the gene for Somatostatin, a 14-residue peptide (Fig. 31.27) found in hypothalamic extracts. Somatostatin inhibits the secretion of Growth Hormone, insulin, and Glucagon. The DNA molecule encoding this peptide was synthesized by joining eight oligonucleotide blocks. This gene was fused to the β-galactosidase gene located in a plasmid vector. Upon Transformation of E. coli, synthesis began of a fusion protein in which somatostatin was linked to β-galactosidase. The peptide bond between these two components was cleaved in vitro using Cyanogen bromide (Section 2.7). To accomplish this, the engineered gene contained a Methionine codon immediately preceding the first codon of somatostatin. The C-terminus of somatostatin was free, as two stop codons were positioned right after the codon for the final peptide residue (Fig. 31.27). The chimeric protein accounted for a significant fraction of the total cellular protein (about 3%). Moreover, the somatostatin obtained in this manner was biologically active. Consequently, cloning a chemically synthesized gene can yield a functionally active polypeptide.

Fig. 31.27. Synthesis of the peptide hormone somatostatin by E. coli cells transformed with a chemically synthesized gene

31.14. Prospects for Gene Cloning

Recombinant DNA technology has opened up new horizons in molecular biology. Amplifying genes through bacterial cloning provides unlimited amounts of DNA for electron microscopic analysis and nucleotide sequencing. Specific DNA regions responsible for gene mobility, DNA Replication, and transcription are now being investigated. Entirely new avenues of research are emerging; a prime example is the discovery of intervening sequences in many eukaryotic genes. It has become possible to rapidly map complex Chromosomes and dissect them into individual elements amenable to various experimental manipulations. The pace of research is constantly accelerating. Furthermore, Gene cloning has become an important method for producing specific Proteins in large quantities. For example, recombinant molecules can be used to increase the yield of DNA ligase produced in E. coli cells by 500-fold. Perhaps the most promising prospect is the synthesis of eukaryotic Peptides and Proteins by transformed bacteria. In the near future, Hormones such as insulin and antiviral agents such as interferon will be produced by bacteria¹. A new era in pharmacology is beginning, which is bound to have a profound impact on medicine. The potential of gene cloning to enhance agricultural production is also being actively explored. Eukaryotic genes are currently being introduced not only into bacteria but also into Eukaryotic cells. For example, SV40 virus has been used as a vector to transfer the rabbit globin gene into monkey Kidney cells. Such infected cells synthesized significant amounts of rabbit β-globin. This experimental approach holds great promise as a method for deciphering The regulatory mechanisms of EUKARYOTIC Gene Expression.

¹ Insulin synthesized by bacterial cells is already on sale in England and the USA. Transl.